Mechanisms of excitation of spinal networks by stimulation of the ventral roots

Abstract

It has recently been demonstrated that motoneurons in neonatal rodents release an excitatory amino acid, in addition to acetylcholine, from their central terminals onto Renshaw cells. Although the function of this amino acid release is not understood, it may mediate the excitatory actions of motor axon stimulation on spinal motor networks. Stimulation of motor axons in the ventral roots or muscle nerves can activate the locomotor central pattern generator or entrain bursting in the disinhibited cord. Both of these effects persist in the presence of cholinergic antagonists and are abolished or diminished by ionotropic and metabotropic glutamate antagonists. Calcium imaging in the disinhibited cord shows that a ventral root stimulus evokes ventrolateral activity initially, which subsequently propagates to the rest of the cord. This finding suggests that excitatory interneurons excited by motoneuron recurrent collaterals are located in this region. However, motoneurons do not exhibit short latency excitatory potentials in response to ventral root stimulation indicating that the excitatory effects are mediated polysynaptically. We discuss the significance of these findings.

Figure 1. Motoneurons release an excitatory amino acid from their terminals onto Renshaw cells in addition to acetylcholine

(A) Schematic showing a whole cell recording from an identified Renshaw cell (blue filled circles) and a suction electrode on the ventral root to antidromically stimulate motoneurons (grey filled circles) in the isolated spinal cord in vitro. (B) Synaptic responses in response to a single antidromic stimulus applied to the ipsilateral ventral root. Addition of cholinergic blockers reduced but did not abolish the response. The response was abolished by the addition of the glutamatergic antagonists APV and CNQX. The data in B were modified from ref .

Figure 2. Locomotor-like activity and disinhibited bursting evoked by stimulation of motor axons exhibit similar properties in the neonatal spinal cord of the mouse

(A) Locomotor-like activity evoked by a train of stimuli applied to the sciatic nerve. To ensure exclusive stimulation of motor axons, the ipsilateral dorsal roots were cut. The records are direct coupled (DC) recordings of the electrical activity recorded from the left (red traces) and right (blue traces) lumbar 1 (L1) ventral roots. The rectangle in this and the subsequent panels highlights the alternation between the activity on the left and right sides. The top set of traces (control) show the activity induced by sciatic nerve stimulation in the absence of drugs. The next traces show that the locomotor-like activity persists in the presence of a cocktail of cholinergic antagonists (50 μM mecamylamine, 50μM dihydro-β-erythroidine, and 5 μM atropine), although the discharge has been reduced. In the presence of the NMDA antagonist APV, the locomotor-like activity is abolished and it recovers when the drugs are washed out (Washout). (B) The phase relations between the activity recorded from the left and right L1 and the right L5 ventral roots under control conditions (control) are unchanged in the presence of cholinergic blockade (chol. block). (C) Spontaneous bursting in the presence of the inhibitory antagonists bicuculline and strychnine can be entrained by a brief train of 5 stimuli (20 Hz, arrowheads) applied to an adjacent ventral root. The effect is maintained in the presence of cholinergic blockade but abolished in the presence of the AMPA/kainate antagonist CNQX (panel D). (E) Histogram comparing the efficacy of ventral root stimulation at evoking locomotor-like activity (open bars) or the entrainment of disinhibited bursting (filled bars). The ordinate shows the percentage of experiments in which the ventral root stimulus produced locomotor-like activity or entrained disinhibited bursting. Modified from refs (A and B) and (C-E).

Figure 3. Ventral root responses evoked in motoneurons exhibit prolonged delays that may reflect metabotropic glutamate receptor activation

(A) Whole cell voltage clamp recording from an identified motoneuron together with the DC extracellular signal recorded from the right L1 ventral root during an episode of locomotor-like activity evoked by a train (4Hz, 10s) of ventral root stimuli. The responses evoked by the first 5 stimuli (delineated by the grey rectangle) were averaged and are displayed in the expanded traces on the right. The arrow marks the onset of the evoked inward current with a latency of 50ms from the ventral root stimulus (red dotted line). (B) During disinhibited bursting entrained by a brief train of stimuli (5 stimuli at 20Hz) applied to an adjacent ventral root the delay between the last stimulus in the train and the onset of the evoked burst (arrows and delay on 4^th burst) progressively increases. The delays are indicated over the individual bursts. (C) Locomotor-like activity evoked by a ventral root stimulus train is reversibly abolished in the presence of the mGluR1 receptor antagonist CPCCOEt. The bars below the records indicate the duration of the stimulus train. (D) Schematic of the ventral part of the cord showing the possible loci of action of CPCCOEt. It is assumed that motoneuron collaterals release glutamate at an excitatory interneuron that is distinct from Renshaw cells. This interneuron in turn projects to the burst generating network. CPCCOEt could act presynaptically on motoneuron synaptic terminals (1), pre- and/or postsynaptically on the excitatory interneuron (2) or within the burst network itself (3). Parts of the figure (B and C) were modified from ref. .

Figure 4. Calcium imaging of the cut face of the disinhibited spinal cord (rostral L5) showing the ventrolateral origin of optical activity following a ventral root stimulus or at the onset of some spontaneous bursts

(A) Schematic of the spinal cord preparation showing the cut face of the rostral L5 segment. One hemi-segment was injected with the calcium-sensitive dye fluo-3 (green region). (B) Series of images showing the ventrolateral origin of activity following a brief stimulus train (5 stimuli, 20Hz) applied to the ipsilateral L5 ventral root. Each image is a difference image obtained by subtracting a pre-stimulus control image from the active frame. The time of acquisition of the frame is indicated above the images. (C) A similar pattern of activation can be observed during ~40% of the spontaneously occurring bursts. Modified from ref. .